March 23, 2016
700 Clark Hall, 12:15
James Weisshaar
University of Wisconsin
Spatiotemporal Organization of
the E. coli Cytoplasm
Superresolution fluorescence microscopy has enabled us to locate and track single ribosomes
(chromosomally expressed S2-mEos2), RNA polymerase copies (chromosomally expressed b’-mEos2),
and DNA loci (ParB-XFP labeling) in live E. coli with spatial accuracy of s ~ 30 nm and time resolution of
2-10 ms when needed. Ribosome-RNAP segregation is strong, arguing against co-transcriptional
translation as the primary means of protein synthesis. Diffusion of both ribosomes and RNAP is
heterogeneous. This enables us to distinguish translating 70S-polysomes from 30S subunits searching for
translation initiation sites. We can also distinguish transcribing RNAP copies from those searching for
transcription initiation sites. Time-dependent imaging of the DNA stain Sytox Orange after drug
treatment indicates that on the 0-5 min timescale, both rifampicin and chloramphenicol induce nucleoid
contraction. This corroborates the transertion hypothesis (co-transcriptional translation and
simultaneous insertion of membrane proteins). The combination of these new experimental data with
coarse-grained models of DNA-ribosome mixing suggests a picture in which expansion of the nucleoid by
transertion is important for optimal cell function. The expanded nucleoid enables facile recycling of
ribosomal subunits from ribosome-rich regions (where most translation occurs) to the nucleoids (where
they can initiate co-transcriptional translation). At the same time, free polysomes are excluded from the
nucleoids. The resulting spatial segregation may enhance overall growth rate by restricting the space
within which RNAP searches for transcription initiation sites and ribosomal subunits search for
translation initiation sites. This in turn may enhance overall growth rate.
Hosts: Peng Chen
Biophysics Colloquium chair: Brian Crane
Biophysics Colloquia website: http://www.biophysics.cornell.edu/seminars